Infection, a leading global cause of infant mortality, disproportionately affects the most premature neonates. The innate and adaptive immune systems of the preterm infant are known to be relatively immature in their rapidity and vigor of response to a microbial challenge and this often results in systemic sepsis and high mortality. However, more recent data suggest that once activated, the inflammatory response of the preterm infant fails to be extinguished as promptly as that of the adult subject and this may result in excessive inflammatory complications of diseases that contribute to neonatal morbidity. Mounting evidence indicates a role for T regulatory (Treg) cells in the modulation of inflammatory mechanisms. Microbial activation of Toll-like receptors (TLRs) may promote Treg cell-mediated suppression of effector cells with inflammatory phenotypes and Treg cells can diminish neutrophil accumulation at inflammatory sites, actions that dampen inflammation. Conversely, alteration of Treg function may promote inflammatory responses. Activation of certain TLRs may reverse Treg cell suppressive activity, and exposure to an inflammatory milieu can induce Treg cell conversion to inflammatory Th1 and/or Th17 cells that also crosstalk with neutrophils, although whether neutrophils are instructive to Treg cells to amplify inflammation is unknown. Thus, the exact mechanisms and conditions that determine whether Treg cells suppress or promote inflammation are incompletely defined. Furthermore, whether interactions between innate and adaptive immune mechanisms contribute to neonatal inflammatory responses in the context of developmental immaturity or infection is unexplored. We hypothesize that neonatal developmental immaturity is associated with compromised adaptive-innate immune interactions that promote rather than suppress inflammatory responses to infection, and further postulate that this imbalance exacerbates inflammation in the most immature neonates. For this Exploratory R21 mechanism, we propose comparative ex vivo studies of human preterm, term and adult immune cells to: 1) Determine the effect of innate immune (TLR) activation on Treg cell suppressive function and phenotype (Aim 1); and 2) Characterize neutrophil-mediated effects on Treg cell function and plasticity (Aim 2). We expect to confirm preliminary observations of differential TLR expression and neutrophil-Treg cell crosstalk in neonates and adults and anticipate enhanced inflammatory effects in preterm neonates, all of which would establish proof-of-concept. These studies form the basis for future investigations to achieve our long-term goal, which is to elucidate immune and inflammatory mechanisms that contribute to neonatal health and disease. This project is specifically responsive to a NIAID Concept focused on neonatal immune responses and thus a research area of high priority. PUBLIC HEALTH RELEVANCE: Premature newborns are at high risk for severe infection, which can lead to inflammation that permanently injures important organs, such as the brain or lungs, or that often causes death. The goal of our studies is to learn how immune cells of premature newborns might interact in abnormal ways that lead to uncontrolled inflammation during severe infection. This new information may help us develop better treatments for premature infants, and in addition may help us understand why other humans with abnormal immune systems, such as those with diabetes or cancer or the elderly, are also at risk for severe infection.